AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H2341-H2348, 2007. First published January 12, 2007; doi:10.1152/ajpheart.01061.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/5/H2341    most recent
01061.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodenwaldt, B.
Right arrow Articles by de Wit, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodenwaldt, B.
Right arrow Articles by de Wit, C.

Endogenous and exogenous NO attenuates conduction of vasoconstrictions along arterioles in the microcirculation

Barbara Rodenwaldt,2 Ulrich Pohl,2 and Cor de Wit1

1Institut für Physiologie, Universität Lübeck, 23538 Lübeck; and 2Physiologisches Institut, Ludwig-Maximilians-Universität Munich, 80336 Munich, Germany

Submitted 27 September 2006 ; accepted in final form 7 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular coordination in the microcirculation depends on gap junctional intercellular communication (GJIC), which is reflected by the conduction of locally initiated vasomotor responses. However, little is known about the regulation of GJIC in vivo. We hypothesized that endothelial NO regulates GJIC and therefore studied whether conduction of constrictions and dilations along the vessel wall is modulated by modifying the level of microcirculatory NO. Arterioles were focally stimulated using high K+ or acetylcholine in the cremaster muscle in situ, and diameter changes were assessed at the local and remote upstream sites by intravital microscopy. Local stimulation with K+ initiated a constriction that conducted along the arteriole with diminishing amplitude (length constant {lambda}: 371 ± 42 µm). After N{omega}-nitro-L-arginine (L-NNA), {lambda} increased to 507 ± 30 µm, indicating that GJIC is attenuated by endogenous NO. Exogenous NO, but not adenosine, reduced {lambda} after L-NNA in a reversible, concentration-dependent, and mainly cGMP-dependent manner as assessed by inhibition of soluble guanylate cyclase. In endothelial NO synthase-deficient mice, {lambda} was 530 ± 80 µm and thus similar to that in wild-type mice after L-NNA. Exogenous NO likewise reduced {lambda} in these mice. The effects of NO were comparable to those of wild-type animals in Cx40-deficient mice, which excludes Cx40 as a specific target of NO. In contrast to constrictions, the amplitude of conducted dilations on acetylcholine did not diminish up to 1,300 µm and were not altered by L-NNA or exogenous NO. We conclude that endogenously released NO attenuates the conduction of vasoconstrictions most likely due to a modulation of gap junctional conductivity. We suggest that this effect is specific for smooth muscle cells, which probably transmit constricting signals, and involves connexins other than Cx40. This mechanism may support the dilatory potency of NO by preventing the conduction of remote vasoconstrictions into areas with basal or activated NO release.

conducted responses; gap junctional communication; connexins; nitric oxide

BLOOD FLOW IS REGULATED to precisely meet the metabolic tissue demands. The adjustment of flow requires a coordination of diameter changes over substantial distances along the vessel length in the microcirculation that is believed to depend on intercellular communication through gap junctions (31). Such a communication along the vessel wall gives rise to coordinated vasomotor responses, e.g., conducted constrictions and dilations, that reflect the synchronous activity of vascular cells. It has been proposed that the mechanism underlying conduction of vasomotor responses entails the spread of membrane potential changes along endothelial and/or vascular smooth muscle cells through gap junctions (9). There is good experimental evidence to support this idea, because hyper- or depolarizations originating at discrete sites after focal stimulation can be demonstrated at remote sites in vascular cells along the vessel length (11, 32, 39). Because gap junctions form intercellular low-resistance channels, they allow the spread of electrotonic signals longitudinally along the vessel through a layer of coupled cells (7).

The well-accepted important functions of gap junctional intercellular communication (GJIC) in the vessel wall contrast with the sparse investigation of the control and regulation of GJIC in arterioles. In cell culture systems GJIC is modulated by different mechanisms that include biosynthesis of the pore-forming connexins (Cx) and their transport and assembly, as well as formation and removal of gap junctions from the cell membrane (3). Especially internalization and degradation may be triggered by phosphorylation of connexins, specifically Cx43 (37). This member of the large family of connexins also is found in vascular gap junctions alongside Cx40, Cx37, and Cx45, suggesting that GJIC also is subject to modulation in vessels. In addition to formation and degradation of gap junctions that may enhance or restrict GJIC at larger time scales, channel permeability and/or conductivity of existing gap junctions underlies regulatory processes as has been demonstrated in a number of different settings. In cells transfected with Cx43 cDNA, cGMP shifted the channel conductance states to lower values that coincided with a change of the phosphorylation status (20), indicating that phosphorylation may represent a mechanism to posttranscriptionally regulate gating properties. Moreover, nitric oxide (NO) reduced permeability and conductivity of gap junctions in a cGMP-dependent fashion in neuronal (25, 30) and cardiac cells (19), although an enhancement of GJIC by NO also has been reported (40). In vascular cells GJIC has been demonstrated to be modulated by inflammatory mediators in vitro (15). In fact, most connexins that are expressed in vascular cells (Cx40, Cx45, Cx43) and form gap junctions are targets of phosphorylation (21, 35, 36). Moreover, inflammatory mediators have been demonstrated to modulate intercellular coupling in arterioles in vivo (34), supposedly in a NO-dependent manner (22, 26). In addition, it is rather unclear which connexin is targeted by such a regulatory mechanisms in vivo. Initially, Cx43 was proposed as a target (23), and later Cx37 (26) and Cx40 during study of hypoxia/reoxygenation (4).

Therefore, we hypothesized that endogenous NO acts as a regulatory factor on GJIC in vivo in a cGMP-dependent fashion. We tested whether inhibition of endogenous NO synthesis alters the conduction of constrictions and/or dilations along the vessel wall. To this end, arterioles in the microcirculation in vivo were locally stimulated to initiate conducted responses in a model of acute and chronic NO deficiency. Moreover, we investigated whether exogenous NO is able to restore a potential effect of endogenous NO and compared the effects to other vasodilators that act independently of the NO/cGMP pathway. Finally, we examined Cx40-deficient mice to evaluate the role of Cx40 as a target of regulation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care and experimental procedures were performed in accordance with the guidelines for the care and use of laboratory animals published by the National Institutes of Health and the German animal protection law as approved by the local government. Male mice (C57BL/6, body weight: 20–40 g, age: 6–10 mo) were anesthetized by intraperitoneal injection of droperidol (20 mg/kg), midazolam (2 mg/kg), and fentanyl (0.1 mg/kg), followed by continuous intravenous infusion. In addition to wild-type (C57BL/6), mice deficient for endothelial NO synthase (eNOS–/–) (13) and Cx40 (Cx40–/–) (17), both in a C57BL/6 genetic background, were investigated. The cremaster muscle was prepared as described (38) and superfused with bicarbonate-buffered salt solution. Arterioles were studied using a microscope (Metallux; Leitz, Wetzlar, Germany) equipped with a video camera. Images were recorded on videotape (S-VHS; Sony) for off-line measurements of luminal diameters after digitization (OPTIMAS; Media Cybernetics, Silver Spring, MD). Images were displayed at 1,000-fold magnification, and the resolution after digitization was ~1.0 µm.

Conducted constrictions or dilations were initiated by locally confined stimulation of arterioles with K+ solution (3 mol/l) or acetylcholine (10 mmol/l) ejected from glass micropipettes (80–140 kPa, 40–400 ms). The ejection time was varied to induce similar local responses between the different treatment groups (see below). Micropipettes had a tip opening of 1–2 µm (Brown-Flaming, model P-97; Sutter) and were positioned in close proximity to the vessel (38). The arteriole was continuously monitored before and after stimulation at the application site (local). If a response at the local site was obtained, the same stimulus was applied again without repositioning the pipette, and the arteriole was observed at distant, upstream sites (335–2,000 µm). This protocol was repeated after superfusion of substances to alter the level of microvascular NO, consisting of blockade of NO synthase (30 µmol/l N{omega}-nitro-L-arginine, L-NNA) and subsequent addition of NO donors (0.03–1 µmol/l sodium-nitroprusside, SNP; 0.1–1 µmol/l S-nitroso-N-acetyl-D,L-penicillamine, SNAP). Thereafter, exogenous NO was washed out, and an NO-independent vasodilator (adenosine) was applied instead. The effects of NO also were studied in the presence of an inhibitor of soluble guanylate cyclase (10 µmol/l 1H-1,2,4-oxadiazolo-4,3-aquinoxalin-1-one, ODQ). In addition to wild-type, eNOS–/– and Cx40–/– animals were studied. One or two arterioles were studied in each experiment in duplicate. Maximal diameters of the arterioles were determined by simultaneous application of different vasodilators (SNP, acetylcholine, and adenosine, each 30 µmol/l). SNAP was obtained from Alexis Biochemicals (Grünberg, Germany); all other substances were obtained from Sigma (Deisenhofen, Germany) except for the salts used to prepare the superfusion (Merck, Darmstadt, Germany). SNP was dissolved in 1 mmol/l Na-acetate and L-NNA (5 mg/ml) in water. All further solutions were prepared in the superfusion buffer.

The change in diameter was normalized to the maximal possible response:

Formula 1(1)
where DSt represents the diameter after stimulation, DCo is the respective control diameter, and DMax is the maximal possible response, which for dilations is the maximal diameter or for constrictions, the minimal luminal diameter (zero). Comparisons within groups were performed using paired t-tests and, for multiple comparisons, corrected according to Bonferroni. Data between groups were compared using analysis of variance (ANOVA) followed by post hoc analysis of the means. Length constants ({lambda}) for conduction were calculated by assuming an exponential decay of the constriction along the vessel. Accordingly, constrictions at upstream sites were normalized to the local constriction, which was set to 100%, and these normalized data were fitted using a nonlinear least-squares method to the equation f(x) = 1/e(x/{lambda}), where x represents the distance along the vessel and calculated {lambda} gives the distance at which the constriction has diminished to 37% (1/e). Differences between treatment groups were assessed by pooling the data and by a comparison of the goodness of fits between separate and pooled data (27). Differences were considered significant at a corrected error probability of P < 0.05. Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Locally initiated vasoconstrictions conduct along the arteriolar wall. Data were obtained in 56 arterioles with a maximal diameter between 40 and 87 µm (58 ± 2 µm) in 38 wild-type mice. The resting tone, i.e., the quotient of resting and maximal diameter, was 0.87 ± 0.01 in these first- and second-order branching arterioles. Brief application of K+ (3 mol/l, 230 ± 55 ms) in the vicinity of the vessel induced a transient constriction, the maximum of which was reached 5 s after K+ application. Therefore, diameter changes 5 s after application were used for all further calculations. At this time point the arterioles constricted from 53 ± 3 to 25 ± 2 µm (–54 ± 3%) at the site of application and thereafter reattained their initial diameters within 20 s (Fig. 1A). This local constriction was conducted to remote upstream sites without measurable delay. However, the amplitude of the constriction decayed with distance along the arteriole. At a distance of 335 µm, arterioles constricted by –31 ± 6%, and at 670 µm, by –11 ± 4%. Significant constrictions (2 ± 2%) were not observed 1,000 µm upstream (Fig. 1B). The calculated {lambda} of the conducting constriction was 371 ± 42 µm in these untreated preparations. A nonspecific blocker of gap junctions, carbenoxolone, was used to verify that constrictions are conducted to remote sites via gap junctions. In the presence of carbenoxolone (50 µmol/l), local K+-induced constrictions remained unaffected (–61 ± 3 vs. –60 ± 8%), but the remote responses were strongly attenuated (335 µm: from –47 ± 12 to –8 ± 6%) or abolished at a further distance (670 µm: from –11 ± 6 to 6 ± 2%, n = 10 arterioles in 4 mice).


Figure 1
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 1. Nitric oxide (NO) attenuates conducted constrictions initiated by local depolarization. Brief focal application of K+ (3 mmol/l) initiated a local vasoconstriction that was conducted to remote upstream sites nearly instantaneously. A: diameter changes as a function of time at different sites along the arteriole (local, 335 µm, 670 µm, and 1,000 µm upstream) during local stimulation with K+ (arrow) in the presence or absence of NO. B: comparison of the diameter change 5 s after stimulation, which represents the maximal constriction. In untreated preparations (Con), constrictions decayed strongly with distance. After N{omega}-nitro-L-arginine (L-NNA; 30 µmol/l), constrictions at remote sites were significantly enhanced, which was reversed after addition of sodium nitroprusside (SNP; 1 µmol/l). Replacing SNP with an NO-independent dilator (adenosine, Ado; 1 µmol/l) enhanced remote constrictions again. Local constrictions were kept at a constant amplitude by adjusting the pressure pulse application of K+. Constrictions are given as percentages of maximal response; n = 6–7 arterioles in 6 experiments. *P < 0.05; **P < 0.01.

 
NO attenuates the conduction of locally initiated vasoconstrictions. Endogenous NO production was inhibited by L-NNA (30 µmol/l) to examine the effect of NO on the conduction of vasoconstrictions. L-NNA induced a slight constriction from 59 ± 3 to 55 ± 4 µm (P = 0.06, Table 1). The locally initiated constriction was kept constant by adjusting the ejection time of K+ (Fig. 1). However, stimulation time was not significantly different from control (control: 284 ± 58 ms, L-NNA: 389 ± 78 ms). Despite a similar local constriction, the responses at upstream sites were enhanced in the absence of endogenous NO. Moreover, after L-NNA, a significant constriction was observed at a distance of 1,000 µm, where diameter changes were not found in untreated preparations. Thus, {lambda} increased from 277 ± 40 to 507 ± 30 µm (P < 0.001) after L-NNA (Fig. 2). Exogenous NO was added by superfusing SNP (1 µmol/l) to test for the reversibility of the effect. The diameter of these large vessels (Table 1) and the K+ stimulation pulse (436 ± 94 ms, P = not significant vs. L-NNA) to elicit a similar local constriction were not different in the presence of SNP compared with L-NNA treatment. Despite a similar local response, constrictions at remote upstream sites were significantly attenuated in the presence of SNP (Fig. 1). The length constant decreased ({lambda} = 240 ± 20 µm, P < 0.001 vs. L-NNA, Fig. 2) and was not different from that of untreated arterioles in this series ({lambda} = 277 ± 40 µm). Because the attenuation of remote constrictions might be related to a nonspecific dilator effect opposing a constriction, we also examined adenosine, which acts by NO/cGMP-independent mechanisms. After washout of SNP and addition of adenosine (1 µmol/l), constrictions at remote sites were again enhanced despite a similar magnitude of the local response (pulse time: 280 ± 89 ms, P = 0.06 vs. SNP, Fig. 1). Thus the length constant increased again back to the value observed before and was significantly higher in the presence of adenosine than in the presence of SNP ({lambda} = 720 ± 65 µm, P < 0.001 vs. SNP, Fig. 2).


View this table:
[in this window]
[in a new window]

  		Table 1. Arteriolar resting and maximal diameter in different treatment groups

 

Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 2. The decay of the constrictions with distance is accelerated by NO. Remote constrictions were normalized to the local response that was initiated by K+ and set to 100%. Assuming an exponential decay of the constriction with distance, the normalized values (circles) were fitted to the equation f(x) = 1/e(x/{lambda}), and the predicted decay is depicted as a function of distance (lines). The point of intersection of the predicted curve with a constriction of 37% (1/e) gives the calculated length constant {lambda}. L-NNA (30 µmol/l) increased {lambda} (from 277 ± 40 to 507 ± 30 µm, P < 0.001), which was reversed by SNP (1 µmol/l, {lambda} = 240 ± 20 µm, P < 0.001 vs. L-NNA). Substitution of SNP by Ado (1 µmol/l) abrogated this effect, and {lambda} increased again (720 ± 65 µm, P < 0.001 vs. SNP). n = 6–7 arterioles in 6 experiments.

 
Concentration-dependent effects on conduction by different NO donors. In this experimental series (n = 7 arterioles in 6 mice), the efficacy of SNP at different concentrations was evaluated. Resting diameters (Table 1) and locally initiated constrictions were similar between different groups (L-NNA: –60 ± 4%, SNP at 30 nmol/l: –60 ± 5%, SNP at 0.1 µmol/l: –59 ± 6%). In the presence of L-NNA, {lambda} was 674 ± 47 µm in this series and was not altered by 30 nmol/l SNP ({lambda} = 640 ± 79 µm). However, 100 nmol/l SNP decreased the length constant significantly ({lambda} = 462 ± 73 µm, P < 0.01 vs. L-NNA). Moreover, in the presence of higher concentrations of SNP (1 µmol/l), the length constant was even shorter ({lambda} = 240 ± 20 µm, P < 0.01 vs. SNP at 100 nmol/l, Fig. 3A). A chemically different NO donor was tested in a subset of experiments (n = 5 arterioles in 4 mice). K+-induced constrictions were not different at the local site (L-NNA: –64 ± 5%, SNAP at 0.1 µmol/l: –61 ± 4%, SNAP at 1 µmol/l: –55 ± 5%) without significant changes of exposure time to K+. However, in the presence of SNAP (1 µmol/l), constrictions at remote sites were significantly attenuated (670 µm: from –20 ± 3 to 4 ± 3%, 1,000 µm: from –9 ± 3 to 4 ± 3%), whereas the lower concentration of SNAP did not alter the responses. Thus {lambda} was reduced by 1 µmol/l SNAP from 480 ± 30 to 310 ± 26 µm (P < 0.01, Fig. 3B) but was not significantly modulated by 0.1 µmol/l SNAP ({lambda} = 426 ± 46 µm). This demonstrates a concentration-dependent effect of SNP and SNAP. However, if endogenous NO production was not blocked, SNP was without effect. The length constant in untreated preparations was 420 ± 50 µm and was not altered after addition of 1 µmol/l SNP ({lambda} = 524 ± 60 µm, n = 6 arterioles in 3 mice).


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Concentration-dependent effect of NO donors. Remote constrictions upon K+ depolarization are normalized to the local response. Normalized constrictions (circles) and predicted decay curves (lines) are depicted as a function of distance from the stimulation site. A: 100 nmol/l SNP reduced {lambda} from 674 ± 47 to 462 ± 73 µm (P < 0.01), but a lower concentration was without effect (30 nmol/l: {lambda} = 640 ± 79 µm). Increasing the concentration of SNP had an additional effect (1 µmol/l: 240 ± 20 µm, P < 0.01 vs. 100 nmol/l SNP). B: the chemically distinct NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 1 µmol/l) also reduced {lambda} (from 480 ± 30 to 310 ± 26 µm, P < 0.01), but {lambda} remained unaltered at the lower concentration of SNAP (0.1 µmol/l: 426 ± 46 µm). SNP: n = 6–7 arterioles in 6 experiments, SNAP: n = 5 arterioles in 4 experiments. All experiments were performed in the presence of L-NNA (30 µmol/l).

 
Effect of NO is partially cGMP dependent. Work in our laboratory (18) has previously shown that the dilation induced by NO donors is mediated by the NO/cGMP/cGMP-dependent kinase (cGKI) pathway. Therefore, we examined whether the modulation of conduction is likewise cGMP dependent by blocking the guanylate cyclase using ODQ. Dilations induced by SNP (1 µmol/l) were abolished in the presence of ODQ (10 µmol/l), showing an efficient blockade of the cGMP pathway (data not shown). All preparations were treated with L-NNA in these experiments (n = 7 arterioles in 7 mice), and the locally induced constrictions were not different between different treatments. In the presence of SNP (1 µmol/l), the constrictions at remote sites were attenuated (670 µm: from –11 ± 3 to –1 ± 2%), and after addition of ODQ, they were enhanced again (–10 ± 3%, P < 0.05 vs. SNP). The length constant that was attenuated by SNP from 394 ± 44 to 190 ± 19 µm (P < 0.05) was significantly increased by ODQ (250 ± 18 µm, Fig. 4). However, the effect of SNP was not completely reversed, and {lambda} was significantly lower compared with that determined in the presence of L-NNA alone.


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 4. Effect of exogenous NO is partially cGMP dependent. Conducted constrictor responses were normalized to the locally initiated K+ response. Normalized constrictions (circles) and predicted decay curves (lines) are depicted as a function of distance from the stimulation site. SNP (1 µmol/l) also reduced {lambda} in this series (L-NNA: 394 ± 44 µm, SNP: 190 ± 19 µm, P < 0.05). Inhibition of the soluble guanylate cyclase by 1H-1,2,4-oxadiazolo-4,3-aquinoxalin-1-one (ODQ; 10 µmol/l) partially reversed this effect ({lambda} = 250 ± 18 µm, P < 0.05). n = 7 arterioles in 7 experiments.

 
NO is also effective in chronic NO deficiency. In eNOS-deficient mice, 17 arterioles (maximal diameter: 53 ± 2 µm, resting tone: 0.77 ± 0.05) were studied in 12 mice. Locally initiated K+ constrictions conducted along the arteriole, and the calculated length constant was 530 ± 80 µm (n = 6 arterioles in 6 mice, Fig. 5), which was significantly higher than the {lambda} value calculated from data of untreated wild-type mice (371 ± 42 µm, P < 0.05) but similar to that of wild-type after L-NNA (475 ± 28 µm). SNP (1 µmol/l) induced a small but insignificant dilation from 37 ± 1 to 42 ± 1 µm (P = 0.06). In the presence of SNP, a similar local constriction (SNP: –64 ± 4%, control: –68 ± 5%) led to attenuated remote constrictions (335 µm: –21 ± 4 vs. –41 ± 4%, 670 µm: –3 ± 2 vs. –21 ± 7%). Thus {lambda} was significantly reduced to 264 ± 20 µm (P < 0.001, Fig. 5). After washout and addition of adenosine (1 µmol/l), remote constrictions were again enhanced (335 µm: –45 ± 8%, 670 µm: –24 ± 5%) and {lambda} increased back to control level (720 ± 126 µm, Fig. 5). In a second group of 6 eNOS-deficient animals, the effect of the dilator acetylcholine was compared with the effect of SNP in the continuous presence of indomethacin (3 µmol/l) to block any possible prostaglandin production. Resting tone under these conditions was 0.67 ± 0.05 (n = 12). K+-induced constrictions at the local site were not different among control (–50 ± 7%), 10 µmol/l acetylcholine (–48 ± 4%), or 1 µmol/l SNP (–41 ± 7%). Whereas acetylcholine did not modulate the constrictions at remote sites (335 µm: –38 ± 6 vs. –32 ± 8%, 670 µm: –21 ± 6 vs. –25 ± 7%), SNP attenuated these constrictions as observed before (335 µm: –13 ± 4%, 670 µm: –9 ± 4%, each P < 0.05 vs. control).


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 5. NO also attenuates the conduction of constriction in endothelial NO synthase (eNOS)-deficient mice. Local constrictions were initiated by K+ depolarization. Remote constrictions (normalized to the local response, circles) and predicted decay curves (lines) are depicted as a function of distance. In eNOS-deficient mice, calculated {lambda} for constrictions was 530 ± 80 µm (Con) and was reduced by SNP (1 µmol/l: 264 ± 20 µm, P < 0.001) without prior addition of L-NNA. Washout of SNP and its substitution by Ado reverted the effect of SNP and induced a rise of {lambda} (720 ± 126 µm, P < 0.001 vs. SNP). n = 6 arterioles in 6 experiments.

 
Similar effect of NO in Cx40-deficient mice. A possible role of Cx40 as the target of the modulatory effect of NO was examined in 11 arterioles (maximal diameter: 48 ± 8 µm) in Cx40-deficient mice. Locally initiated constrictions (–50 ± 3%) conducted to remote sites in untreated preparations (335 µm: –8 ± 2%, 670 µm: 2 ± 2%). The conduction was enhanced after L-NNA (335 µm: –21 ± 4%, P < 0.01, 670 µm: –7 ± 3%, P = 0.08), although the local constriction was similar (–55 ± 4%). After addition of SNP (1 µmol/l), remote constrictions were attenuated again (335 µm: –8 ± 2%, P < 0.01 vs. L-NNA, 670 µm: 0 ± 2%) despite a similar local response (–54 ± 4%). However, after replacement of SNP by adenosine (1 µmol/l), conducted responses were re-enhanced (335 µm: –19 ± 2%, P < 0.01 vs. SNP, 670 µm: –3 ± 2%) despite a similar magnitude of the local constriction (–58 ± 4%). Thus {lambda} increased significantly after L-NNA (from 151 ± 18 to 295 ± 32 µm, P < 0.001), was reduced again by SNP (161 ± 11 µm, P < 0.001 vs. L-NNA), and was re-enhanced after its replacement by adenosine (251 ± 27 µm, P < 0.001 vs. SNP).

Conducted dilations remain unaltered by NO. Finally, a possible modulation of NO on the conduction of locally initiated dilations was examined. Local acetylcholine application (10 mmol/l) induced a dilation in untreated wild-type mice that conducted to remote sites (Fig. 6A). The maximal dilation of 53 ± 5% occurred at ~10 s after acetylcholine application (n = 6 vessels in 6 mice, maximal diameter: 37 ± 2 µm). The amplitude of the dilation decayed only slightly up to a distance of 2,000 µm (1,340 µm: 43 ± 3%, 2,000 µm: 27 ± 10%), which rendered the calculation of {lambda} impossible. Inhibition of endogenous NO production by L-NNA did not modify the amplitude of the dilation at local and remote sites (local: 44 ± 5%, 1,340 µm: 39 ± 7%, 2,000 µm: 27 ± 10%). Also, the temporal behavior of the dilation was mostly unchanged except for a slight shortening of the response at the local site (Fig. 6A). The effect of exogenous NO was studied in seven arterioles in six eNOS–/– mice (maximal diameter: 49 ± 3 µm). Local and remote dilations on acetylcholine were similar to responses observed in wild-type animals (Fig. 6B). The maximal amplitude of the dilation did not decay up to 2,000 µm (local: 45 ± 4%, 670 µm: 47 ± 2%, 1,340 µm: 42 ± 4%, 2,000 µm: 46 ± 4%). In the presence of SNP (30 nmol/l), dilatory amplitudes remained unaltered at all locations studied (local: 42 ± 3%, 670 µm: 48 ± 3%, 1,340 µm: 42 ± 5%, 2,000 µm: 38 ± 6%), and also, the temporal behavior of the responses was unchanged (Fig. 6B).


Figure 6
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 6. The conduction of acetylcholine (ACh)-induced dilations is not modulated by NO. Dilations initiated by local application of ACh (arrows) conducted rapidly along the arterioles to remote sites (observed at 670, 1,340, and 2,000 µm upstream). The maximal amplitude of the dilation was reached within 10 s at all sites and diminished only marginally up to a distance of 2,000 µm in wild-type (A; n = 6 arterioles in 6 experiments) and eNOS-deficient (eNOS–/–) mice (B; n = 7 arterioles in 6 experiments) in untreated preparations (Con). Inhibition of endogenous NO production (L-NNA, 30 mmol/l) in wild type (A) was without effect on the dilatory amplitude at the local and remote sites. Conversely, addition of exogenous NO in eNOS–/– mice (SNP, 30 nmol/l) also was without effect on local and remote dilation. Dilations are given as percentages of the maximal response.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A coordination of cellular behavior in arterioles is most likely achieved via communication through gap junction channels, which is reflected by the conduction of vasomotor signals upon focal stimulation of arterioles. In the present study, we have demonstrated that the conduction of locally initiated constrictions along the vascular wall is regulated by endothelium-derived NO. After inhibition of NO production, the signal eliciting vasoconstriction traveled larger distances along the vessel, which can be reverted by exogenous NO. Since NO-independent vasodilators were without effect, we conclude that NO specifically attenuated the conduction of vasoconstrictions and suggest that NO inhibits gap junctional communication within the vessel wall. The attenuation was partially cGMP dependent. Since NO likewise modulated the responses in Cx40-deficient animals but did not affect the conduction of locally initiated endothelium-dependent dilations, we propose that NO acts on connexins other than Cx40 that are most likely interconnecting the cells of the vascular smooth muscle cell layer.

Local application of K+ at a high concentration induces a local depolarization leading to a constriction, most likely, by activation of voltage-sensitive Ca2+ channels in smooth muscle cells. This response conducts rapidly with a declining amplitude along the vessel. Previous work in our laboratory (8) excluded the possibility that the local constriction is due to an osmotic effect, since application of equimolar Na+ did not change arteriolar diameter, or that the conducting response is due to a release of vasoconstrictors from adrenergic nerves. In fact, the nonspecific gap junction blocker carbenoxolone (6) nearly abrogated remote responses without altering the local constriction, demonstrating that gap junctional communication is a key mechanism to transmit the vasomotor signal along the vessel. The decreasing amplitude of the constriction suggests that the signal declines during the conduction process, which is consistent with passive electrotonic spread of the signal along the vessel wall. Accordingly, from normalized constrictions we calculated the length constant that gives the distance at which the constriction has diminished to 37% of the initial value under the assumption of an exponential decay. After inhibition of NO synthase, the length constant increased significantly, reflecting the enhanced constrictions at remote sites without a change in the local constriction. This suggests that the basal level of endogenously released NO alters the conduction of constrictions along the vessel wall, supposedly by modulation of gap junction conductivity. The readdition of exogenous NO reversed this effect completely within <30 min after its addition, suggesting a rapid regulation of gap junctional communication. This effect was specific for NO, because adenosine that was added instead of a NO donor did not reduce the length constants. Moreover, these data demonstrate the rapid reversibility of the NO effect after inhibition of NO synthase and after washout of the NO donor, which is consistent with an effect on gap junction conductivity rather than an enhanced translocation of additional connexins to the membrane. Conversely, the rapid effect of NO is likewise consistent with a regulation of gap junction conductivity (1) rather than an enhanced degradation of connexins, because the half-life of connexins is ~1.5–5 h (28).

The effect of NO donors was concentration dependent and elicited by two chemically distinct substances. Interestingly, NO was only effective after inhibition of the endogenous NO release. The source of endogenous NO is most likely the endothelial NO synthase, as suggested by two observations in the present study. First, the length constant of the conduction of constrictions in eNOS-deficient mice was similar to that in wild-type mice after inhibition of NO synthase. Second, the NO donor attenuated the conduction in eNOS-deficient mice without prior application of an inhibitor of NO synthase, in contrast to wild-type animals. This suggests that endogenous NO derived from the endothelium or the surrounding skeletal muscle exerts a near-maximal effect on gap junction conductivity. However, under pathophysiological conditions with supposedly even higher NO levels a further attenuation seems to be possible, since treatment with LPS (33, 34) or induction of sepsis attenuated the conduction process in vivo without prior inhibition of endogenous NO release (22, 34). These authors demonstrated that the effect of LPS was mediated by NO and have identified neuronal NO synthase as the source of NO under these conditions (26). Nevertheless, these observations are in good agreement with the data presented in this report, and the present study extends these findings by demonstrating a role for the basal NO release from eNOS and thus expanding the regulatory role for NO toward physiological conditions. Moreover, the partial neutralization of the NO effect by blockade of the soluble guanylate cyclase suggests that the inhibitory effect is mostly mediated by cGMP and, possibly, a subsequent activation of cGKI. Our group (18) has previously demonstrated that the cGKI is an important effector of the NO/cGMP/cGKI pathway in these vessels, and we therefore suggest that cGKI is also involved in the modulation of gap junction conductivity by NO.

In addition to the modulation of gap junction conductivity, the conduction process may have been affected by an effect on cell membrane resistance itself. Activation of a large number of channels in the cell membrane should lead to a stabilization of the actual membrane potential, enhance leak currents through the cell membrane, and, thus, decrease the electrotonic conduction of a remotely initiated potential change along the vessel wall. To test for such a mechanism, we superfused acetylcholine, which induces a hyperpolarization in endothelial and smooth muscle cells by activation of K+ channels (32). These experiments were performed in eNOS-deficient mice to prevent any NO release by acetylcholine. However, acetylcholine did not alter the conduction of vasoconstrictions, whereas in marked contrast, NO reduced in exactly the same preparations again the length constant and remote constrictions. Therefore, we conclude that the effect of NO is most likely due to a modulation of gap junction conductivity and is not caused by an alteration of the electrophysiological behavior of the cell membrane. Because NO acted in a time range of <30 min and its effect was rapidly reversible (20 min), we propose that it acts by modulation of individual channel conductivity and not by a degradation of existing gap junction channels. We excluded Cx40 as the specific target of the modulatory effect of NO because the conduction was also enhanced after inhibition of NO synthase and attenuated again by exogenous NO in Cx40-deficient animals. Because Cx40 is mainly expressed in the endothelium in these vessels (8, 12), we speculate that the smooth muscle layer transmits the signal that leads to remote constrictions, which is supported by the fact that Cx40 deficiency does not alter the conduction of constrictions (8).

In contrast to the conduction of constrictions, the spread of locally initiated dilations was not modulated by NO. Inhibition of NO synthase did not reduce local or remote acetylcholine dilations. This suggests that NO does not contribute to the initiation or the conduction process in this preparation as was observed similarly in the hamster cremaster (14), but NO may contribute in other tissues (5). Conversely, NO synthase inhibition also did not enhance the conduction of dilations in wild-type mice. However, the amplitude of conducted dilations diminished only slightly up to a distance of 2 mm, and an increase after NO synthase inhibition may not have been detectable up to this distance. Larger distances, however, cannot be studied, because the length of the arterioles in the cremaster is limited. The reverse approach, i.e., addition of NO, was tested in eNOS-deficient animals. However, the addition of 30 nmol/l SNP did not reduce the amplitude at the most distant site studied (2 mm). Because the amplitude of the response did not change substantially over this distance, it is not possible to calculate derived parameters and compare length constants. Taking these results together, we could not verify an effect of NO on the process of conduction of dilations as reported after histamine stimulation (29), although using the present approach, we cannot rule out that NO indeed affects it. Previously, we and others proposed that dilatory signals conduct along the endothelium, whereas signals eliciting vasoconstriction conduct along the smooth muscle in the cremaster of mice (2, 8, 24), and modeling likewise suggests distinct conduction pathways (10). The lacking effect of NO on conducted dilations may be due to an amplification mechanism present only in endothelium that is overriding a modulatory effect on gap junction conductivity. This view is supported by the larger distance that is covered by dilations. In addition, different connexins may be involved to interconnect the endothelial and the smooth muscle layer among each other, and these might underlie different regulatory pathways. In vitro data suggest that Cx are differentially regulated by NO. Cx37 or Cx43 have been identified as a target for regulation by NO (16, 19, 40), whereas Cx40 has been shown to be regulated by a cAMP-dependent pathway (35).

In summary, we have demonstrated that NO attenuates the conduction of locally initiated constrictions along the arteriolar wall and suggest that this is due to a modulation of gap junction conductivity. This mechanism may support the dilatory effect of NO by preventing the conduction of remote vasoconstriction into areas with basal or activated NO release. We suggest that this effect is specific for NO and, more importantly, specific for a conducting pathway that physiologically transmits signals eliciting constriction, whereas the dilatory signal conduction pathway along the endothelium remains unaffected. Whether unhindered conduction of vasoconstrictions is contributing to enhanced vascular tone and vascular resistance in conditions with compromised NO function needs to be elucidated in further studies. We excluded Cx40 as a specific target of regulation by NO and propose that the conductivity of gap junctions composed of Cx43 and/or Cx45 is attenuated by NO, because these connexins are expressed in smooth muscle cells.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Deutsche Forschungsgemeinschaft (WI 2071/1-1).


   ACKNOWLEDGMENTS
 
We thank Dr. Axel Gödecke (Zentrum für Physiologie, Düsseldorf, Germany) for kindly supplying eNOS-deficient mice.

Present address of B. Rodenwaldt: Pharmacelsus GmbH, Science Park 2, 66123 Saarbrücken, Germany.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. de Wit, Physiologisches Institut, Universität Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany (e-mail: dewit{at}uni-luebeck.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bao X, Reuss L, Altenberg GA. Regulation of purified and reconstituted connexin 43 hemichannels by protein kinase C-mediated phosphorylation of serine 368. J Biol Chem 279: 20058–20066, 2004.[Abstract/Free Full Text]
  2. Bartlett IS, Segal SS. Resolution of smooth muscle and endothelial pathways for conduction along hamster cheek pouch arterioles. Am J Physiol Heart Circ Physiol 278: H604–H612, 2000.[Abstract/Free Full Text]
  3. Berthoud VM, Minogue PJ, Laing JG, Beyer EC. Pathways for degradation of connexins and gap junctions. Cardiovasc Res 62: 256–267, 2004.[Abstract/Free Full Text]
  4. Bolon ML, Ouellette Y, Li F, Tyml K. Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild-type but not connexin40 null mice in oxidant- and PKA-dependent manner. FASEB J 19: 1725–1727, 2005.[Abstract/Free Full Text]
  5. Budel S, Bartlett IS, Segal SS. Homocellular conduction along endothelium and smooth muscle of arterioles in hamster cheek pouch: unmasking an NO wave. Circ Res 93: 61–68, 2003.[Abstract/Free Full Text]
  6. De Groot JR, Veenstra T, Verkerk AO, Wilders R, Smits JPP, Wilms-Schopman FJG, Wiegerinck RF, Bourier J, Belterman CNW, Coronel R, Verheijck EE. Conduction slowing by the gap junctional uncoupler carbenoxolone. Cardiovasc Res 60: 288–297, 2003.[Abstract/Free Full Text]
  7. De Wit C, Hoepfl B, Wolfle SE. Endothelial mediators and communication through vascular gap junctions. Biol Chem 387: 3–9, 2006.[CrossRef][ISI][Medline]
  8. De Wit C, Roos F, Bolz SS, Kirchhoff S, Kruger O, Willecke K, Pohl U. Impaired conduction of vasodilation along arterioles in connexin40 deficient mice. Circ Res 86: 649–655, 2000.[Abstract/Free Full Text]
  9. De Wit C. Connexins pave the way for vascular communication. News Physiol Sci 19: 148–153, 2004.[Abstract/Free Full Text]
  10. Diep HK, Vigmond EJ, Segal SS, Welsh DG. Defining electrical communication in skeletal muscle resistance arteries: a computational approach. J Physiol 568: 267–281, 2005.[Abstract/Free Full Text]
  11. Emerson GG, Segal SS. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries. Role in vasomotor control. Circ Res 87: 474–479, 2000.[Abstract/Free Full Text]
  12. Figueroa XF, Paul DL, Simon AM, Goodenough DA, Day KH, Damon DN, Duling BR. Central role of connexin40 in the propagation of electrically activated vasodilation in mouse cremasteric arterioles in vivo. Circ Res 92: 793–800, 2003.[Abstract/Free Full Text]
  13. Gödecke A, Decking UK, Ding Z, Hirchenhain J, Bidmon HJ, Gödecke S, Schrader J. Coronary hemodynamics in endothelial NO synthase knockout mice. Circ Res 82: 186–194, 1998.[Abstract/Free Full Text]
  14. Hoepfl B, Rodenwaldt B, Pohl U, de Wit C. EDHF, but not NO or prostaglandins, is critical to evoke a conducted dilation upon acetylcholine in hamster arterioles. Am J Physiol Heart Circ Physiol 283: H996–H1004, 2002.[Abstract/Free Full Text]
  15. Hu J, Cotgreave IA. Differential regulation of gap junctions by proinflammatory mediators in vitro. J Clin Invest 99: 2312–2316, 1997.[ISI][Medline]
  16. Kameritsch P, Khandoga N, Nagel W, Hundhausen C, Lidington D, Pohl U. Nitric oxide specifically reduces the permeability of Cx37-containing gap junctions to small molecules. J Cell Physiol 203: 233–242, 2005.[CrossRef][ISI][Medline]
  17. Kirchhoff S, Nelles E, Hagendorff A, Kruger O, Traub O, Willecke K. Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40-deficient mice. Curr Biol 8: 299–302, 1998.[CrossRef][ISI][Medline]
  18. Koeppen M, Feil R, Siegl D, Feil S, Hofmann F, Pohl U, de Wit C. cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo. Hypertension 44: 952–955, 2004.[Abstract/Free Full Text]
  19. Kwak BR, Jongsma HJ. Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions. Mol Cell Biochem 157: 93–99, 1996.[CrossRef][ISI][Medline]
  20. Kwak BR, Hermans MM, De Jonge HR, Lohmann SM, Jongsma HJ, Chanson M. Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions. Mol Biol Cell 6: 1707–1719, 1995.[Abstract]
  21. Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG, Lau AF. Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. J Cell Biol 149: 1503–1512, 2000.[Abstract/Free Full Text]
  22. Lidington D, Ouellette Y, Li F, Tyml K. Conducted vasoconstriction is reduced in a mouse model of sepsis. J Vasc Res 40: 149–158, 2003.[CrossRef][ISI][Medline]
  23. Lidington D, Tyml K, Ouellette Y. Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43. J Cell Physiol 193: 373–379, 2002.[CrossRef][ISI][Medline]
  24. Looft-Wilson RC, Payne GW, Segal SS. Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle. J Appl Physiol 97: 1152–1158, 2004.[Abstract/Free Full Text]
  25. Lu C, McMahon DG. Modulation of hybrid bass retinal gap junctional channel gating by nitric oxide. J Physiol 499: 689–699, 1997.[Abstract/Free Full Text]
  26. McKinnon RL, Lidington D, Bolon M, Ouellette Y, Kidder GM, Tyml K. Reduced arteriolar conducted vasoconstriction in septic mouse cremaster muscle is mediated by nNOS-derived NO. Cardiovasc Res 69: 236–244, 2006.[Abstract/Free Full Text]
  27. Motulsky HJ, Ransnas LA. Fitting curves to data using nonlinear regression: a practical and nonmathematical review. FASEB J 1: 365–374, 1987.[Abstract]
  28. Musil LS, Le AC, VanSlyke JK, Roberts LM. Regulation of connexin degradation as a mechanism to increase gap junction assembly and function. J Biol Chem 275: 25207–25215, 2000.[Abstract/Free Full Text]
  29. Payne GW, Madri JA, Sessa WC, Segal SS. Histamine inhibits conducted vasodilation through endothelium-derived NO production in arterioles of mouse skeletal muscle. FASEB J 18: 280–286, 2004.[Abstract/Free Full Text]
  30. Rorig B, Sutor B. Nitric oxide-stimulated increase in intracellular cGMP modulates gap junction coupling in rat neocortex. Neuroreport 7: 569–572, 1996.[ISI][Medline]
  31. Segal SS. Regulation of blood flow in the microcirculation. Microcirculation 12: 33–45, 2005.[ISI][Medline]
  32. Siegl D, Koeppen M, Wolfle SE, Pohl U, de Wit C. Myoendothelial coupling is not prominent in arterioles within the mouse cremaster microcirculation in vivo. Circ Res 97: 781–788, 2005.[Abstract/Free Full Text]
  33. Simon AM, McWhorter AR, Chen H, Jackson CL, Ouellette Y. Decreased intercellular communication and connexin expression in mouse aortic endothelium during lipopolysaccharide-induced inflammation. J Vasc Res 41: 323–333, 2004.[CrossRef][ISI][Medline]
  34. Tyml K, Wang X, Lidington D, Ouellette Y. Lipopolysaccharide reduces intercellular coupling in vitro and arteriolar conducted response in vivo. Am J Physiol Heart Circ Physiol 281: H1397–H1406, 2001.[Abstract/Free Full Text]
  35. Van Rijen HV, van Veen TA, Hermans MM, Jongsma HJ. Human connexin40 gap junction channels are modulated by cAMP. Cardiovasc Res 45: 941–951, 2000.[Abstract/Free Full Text]
  36. Van Veen TA, van Rijen HV, Jongsma HJ. Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation. Cardiovasc Res 46: 496–510, 2000.[Abstract/Free Full Text]
  37. Warn-Cramer BJ, Cottrell GT, Burt JM, Lau AF. Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase. J Biol Chem 273: 9188–9196, 1998.[Abstract/Free Full Text]
  38. Wolfle SE, de Wit C. Intact endothelium-dependent dilation and conducted responses in resistance vessels of hypercholesterolemic mice in vivo. J Vasc Res 42: 475–482, 2005.[CrossRef][ISI][Medline]
  39. Yamamoto Y, Klemm MF, Edwards FR, Suzuki H. Intercellular electrical communication among smooth muscle and endothelial cells in guinea-pig mesenteric arterioles. J Physiol 535: 181–195, 2001.[Abstract/Free Full Text]
  40. Yao J, Hiramatsu N, Zhu Y, Morioka T, Takeda M, Oite T, Kitamura M. Nitric oxide-mediated regulation of connexin43 expression and gap junctional intercellular communication in mesangial cells. J Am Soc Nephrol 16: 58–67, 2005.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/5/H2341    most recent
01061.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodenwaldt, B.
Right arrow Articles by de Wit, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodenwaldt, B.
Right arrow Articles by de Wit, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.